Evidence for an increased level of DNA damage in high doubling level human diploid cells in culture

Abstract

Excision repair after ultraviolet (UV) irradiation was estimated in human diploid fibroblast-like cells (HDF) by treating DNA with a crude extract from Micrococcus luteus that contained pyrimidine dimer-specific endonucleases. The assays were done before and after the cells had been arrested in an essentially nonmitotic state. When low and high population doubling level (PDL) cells were assayed under these conditions, no age-related difference in the number of UV-induced endonuclease sensitive sites was observed, but the removal of these sites was more rapid in high PDL arrested cells. There was a difference between the calculated number average molecular weights ( M n) of DNA from unirradiated low and high PDL arrested cells. The lower M n of the DNA from high PDL cells indicated that other enzymes present in the M. luteus extract were acting upon non-UV-induced DNA distortions and that these were present to a greater extent in the chromatin associated regions of older cells. These results support the hypothesis that DNA damage accumulates as HDF progress through their in vitro life span.